The broad long-term objective of the proposed research is to understand in molecular detail the general mechanisms regulating tissue-specific gene expression. This will be accomplished through the study of the human gene encoding glycogen debranching enzyme, and molecular analysis of various subtypes of Type III glycogen storage disease. In this disease there are patients with deficient debranching enzyme activity in both liver and muscle, and patients with deficiency confined to the liver, yet the enzyme is a monomeric protein and appears to be identical in all the tissues. In addition, there are patients with selective loss of one of the two enzyme activities associated with the debranching enzyme, as well as patients with combined deficiency. Experiments with cloning and sequencing of the debrancher gene have suggested that liver and muscle debrancher isoform mRNAs are likely to arise from a single gene through a process of alternative pre-mRNA splicing. The availability of the cloned debrancher gene and nucleotide sequence now allows us to dissect the disease at the molecular level, study the structure-function relationship of this multifunction enzyme, and elucidate mechanisms of gene regulation. The first aim is to further characterize the debrancher isoform mRNAs by determining the partial amino acid sequence from purified liver debrancher protein, and by studying tissue distribution of debrancher isoform mRNAs. The second aim is to determine the structure organization and regulatory regions of the debrancher gene as well as perform functional analysis of the regulatory elements. The third aim is to obtain a complete understanding of the molecular basis of Type III glycogen storage disease by biochemical subtyping of the disease and by identifying mutations causing defective debranching enzyme activity in each subtype. Finally, the structure-function relationship of the debranching enzyme will be characterized by limited proteolysis and mutational analysis of patients with isolated deficiency in one of the two activities associated with the debranching enzyme. Information gained by analysis of the debrancher gene and molecular dissection of different subtypes of Type III glycogen storage disease will provide insight into the molecular basis of the disease, functional domain for this multifunctional enzyme, and general mechanisms controlling tissue-specific gene expression.